Currently, bacteria that may be causing an infection or other health problem are identified by bacteria culture methods. Generally, it takes a day or several days to grow sufficient bacteria to enable the detection and identification of the bacteria. By that time, the person or persons infected by the bacteria may be very sick, or even dead. Thus, there is a need for more rapid detection and identification of bacteria. Further, when bacteria infection is suspected, a physician will often prescribe a broad spectrum antibiotic. This has led to the development of antibiotic resistant bacteria, which has further enhanced the need for more rapid detection of bacteria.
Bacteriophage-based methods have been suggested as a method to accelerate microorganism identification. Bacteriophage are viruses that have evolved in nature to use bacteria as a means of replicating themselves. A bacteriophage (or phage) does this by attaching itself to a bacterium and injecting its genetic material into that bacterium, inducing it to replicate the phage from tens to thousands of times. Some bacteriophage, called lytic bacteriophage, rupture the host bacterium releasing the progeny phage into the environment to seek out other bacteria. Thus, because of the sheer number of the bacteriophage after amplification, in principle it should be easier to detect the bacteriophage than to detect the bacteria. If, in addition the bacteriophage is specific to the bacteria, that is, if the bacteriophage amplification of a particular bacteriophage only occurs for specific bacteria, then the presence of amplified bacteria is then also an indication of the presence of the bacteria to which it is specific. Further, since the total incubation time for infection of a bacterium by parent phage, phage multiplication (amplification) in the bacterium to produce progeny phage, and release of the progeny phage after lysis can take as little as an hour depending on the phage, the bacterium, and the environmental conditions, in principle, bacteriophage amplification can result in much faster detection of bacteria. See, for example, U.S. Pat. No. 5,985,596 issued Nov. 16, 1999 and No. 6,461,833 B1 issued Oct. 8, both to Stuart Mark Wilson; and Angelo J. Madonna, Sheila VanCuyk and Kent J. Voorhees, “Detection Of Esherichia Colil Using Immunomagnetic Separation And Bacteriophage Amplification Coupled With Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry”, Wiley InterScience, DOI:10.1002/rem.900, 24 Dec. 2002, which references are hereby incorporated by reference to the same extent as though fully disclosed herein.
In each of the methods of the above references, samples potentially containing target bacteria are incubated with bacteriophage, as specific as possible for those bacteria. In the presence of the bacteria, the bacteriophage infect the bacteria and replicate in the bacteria resulting in the production of a measurable signal indicating the presence of the target bacteria. Some methods utilize the detection of progeny phage released from infected target bacteria as a means of detection and identification. In this case, progeny phage are not produced if the parent phage do not successfully infect the target bacteria. The degree to which the phage will infect the bacteria if the phage and bacteria are in the same sample is called the infectious sensitivity of the phage. Still other methods rely on the detection of phage replication products rather than whole progeny phage. For example, luciferase reporter bacteriophage produce luciferase when they successfully infect target bacteria. The luciferase then produces light that, if detected, indicates the presence of target bacteria in the sample. The promise of these methods has lead to much research on bacteriophage-based identification of microorganisms. However, as of this writing, no commercially successful method of bacteriophage-based identification has been developed.
In any method based on phage amplification, it is necessary to separate the signal that arises from the parent bacteriophage from the signal from the progeny bacteriophage. U.S. Pat. No. 5,498,525 issued Mar. 12, 1996 to Rees et al. solves this problem by destroying, removing, neutralizing, or inactivating the parent bacteriophage; and U.S. Pat. No. 7,166,425 issued Jan. 23, 2007 to Madonna et al. solves this problem by using a quantity of parent bacteriophage that is below the detection limit of the detection technology. However, to be sure that a lower level of bacteria are detected, the quantity of bacteriophage is kept as high as possible while still being under the detection limit.
While, in principle, bacteriophage-based identification of bacteria should work, commercial success of a bacteriophage-based assay has been impeded by practical problems. In practice, available bacteriophage are not selective to a single strain of bacteria or are not adequately sensitive to the bacterial strain it is desired to detect. Moreover, in the real world, any sample of bacteriophage will contain not only the bacteriophage that are selective of the target bacteria, but other bacteriophage that are selective of non-target bacteria. As a result, in practice, the bacteriophage-based bacteria detection process does not produce a large enough signal in a short enough time to be competitive with bacteria culture methods. We shall refer to this as “assay sensitivity” to differentiate it from infectious sensitivity. Clearly, it would be highly desirable if a bacteriophage process could be provided that had increased selectivity, increased infectious sensitivity, and/or increased test sensitivity and still retained the fast detection of bacteria that is the promise of bacteriophage amplification methods, the potential of which has been driving research in this field.